Diagnostic assays are routinely used to detect the presence and/or quantity of analytes in test samples taken from a patient or other subject. Typical analytes include antigens and antibodies, which are measured using immunodiagnostic techniques to identify certain disease states and various types of non-disease conditions, such as pregnancy. The achievement of high levels of sensitivity and specificity is important in most diagnostic assays. This is particularly true where the analytes of interest are present at relatively low concentrations. Various improvements which have enabled the attainment of higher levels of sensitivity in immunodiagnostic procedures have included the use of monoclonal antibodies in the assay configurations and the incorporation of methods for amplifying the signal used as a tag in these types of assays.
More recently, advances in the field of molecular biology have enabled the detection of specific nucleic acid sequences in test samples using a technique known as probe diagnostics. In probe diagnostics, a nucleic acid sequence is used to "probe" the sample by specifically binding to its complementary nucleic acid target analyte. This makes it possible to detect diseases at an early stage, because the nucleic acid genetic material often appears in a test sample months, or even years, before sufficient time has elapsed for the nucleic acid target to be transcribed into an antigen. This is particularly true in certain types of sexually transmitted diseases, such as infection by the human immunodeficiency virus. Ranki et al, The Lancet, 85(59) 589-593 (1987). In addition to the detection of various diseases and genetic disorders, the ability of probe diagnostics to identify the presence of specific genes can also be used to obtain other pertinent genetic information, such as the presence of genes coding for antigens responsible for graft rejection, as well as genetic information used in cancer and oncogene testing and in forensic medicine.
At its full potential, probe diagnostics are theoretically capable of detecting as little as one molecule in a test sample. One of the major obstacles to achieving the full potential of probe diagnostics is the inherent difficulty which is encountered in detecting the extremely minute quantities of target sequences often present in test samples. As a consequence, recent efforts to improve the sensitivity of probe diagnostics have centered around methods for amplifying the target nucleic acid sequence. Amplification of the target sequence may be accomplished in any one of a number of ways involving the repetitive reproduction or replication of the given DNA or RNA target nucleic acid sequence, resulting in linear or exponential amplification, depending upon the particular method used.
Early methods which were used routinely for production of multiple copies of nucleic acid sequences involved cloning the target nucleic acid sequence into an appropriate host cell system. These methods employ traditional cloning techniques wherein the desired nucleic acid is inserted into an appropriate vector which is subsequently used to transform the host. When the host is grown in culture, the vector is replicated, producing additional copies of the desired nucleic acid sequence. The target nucleic acid sequence which is inserted into the vector can be either naturally occurring or synthesized. In other words, the desired target nucleic acid sequence can be synthesized in vitro and then inserted into a vector which is amplified by growth, as disclosed in U.S. Pat. No. 4,293,652.
U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose an automat able method, commonly referred to as polymerase chain reaction (PCR), for amplifying the amount of target nucleic acid sequence in a test sample. PCR amplification utilizes two oligonucleotide primers which are complementary to the ends of different portions on opposite strands of a section of the target sequence. Following hybridization of these primers to the target, extension products complementary to the target sequence are formed in the presence of DNA polymerase and an excess of nucleoside triphosphates. The primers are oriented so that DNA synthesis by the polymerase proceeds across and through the region between the primers. The hybridized extension product is then denatured from the target and the cycle repeated, with extension product also acting as template for the formation of additional extension product in subsequent cycles of amplification. Cycling continues until a sufficient quantity of the target nucleic acid sequence is produced to result in measurable signal in the assay of choice. Each successive cycle theoretically doubles the amount of nucleic acid synthesized in the previous cycle, resulting in exponential accumulation of amplified product.
International Publication No. WO 89/12696 discloses a different type of automat able amplification procedure. In this type of amplification procedure, presynthesized pairs of amplification probes hybridize contiguously to a section of the target sequence. The contiguous ends are then ligated to form the complementary amplification product. Following ligation, the completed amplification product is separated from the target by heat denaturation. The process is then repeated, with both the target and resulting amplification product acting as a template for the probes in subsequent cycles, until a sufficient quantity of the target nucleic acid sequence is produced to result in measurable signal in the selected assay. As with PCR, each successive cycle theoretically doubles the amount of nucleic acid from a previous cycle. Amplification methods employing presynthesized probes have generally been referred to as ligase chain reaction (LCR), although ligation of the probes can be achieved by means other than the action of a ligase, such as, for example, a chemical or photochemical ligation.
Although nucleic acid amplification methods have revolutionized probe diagnostics by enabling the detection of extremely small quantities of nucleic acid sequences in test samples, they have also created their own problem in the routine diagnostic setting, namely, one of false positives due to carryover contamination. The repeated amplification of a nucleic acid analyte to many millions or billions of times its normal concentration in a test sample raises the possibility of carryover contamination of new samples from the samples containing amplified target. This, in turn, can create artificially high signals in subsequent test samples, including false positives where negative samples are contaminated.
Carryover contamination may occur as the result of mechanical carryover from sample to sample or as the result of airborne contamination. Airborne contamination is unavoidable where reaction vessels containing amplified test sample are opened for any reason, such as for the addition of reagents or for sampling of the amplified analyte for detection. This act alone can aspirate millions of molecules of amplification product into the air, contaminating a normal laboratory work area with hundreds of molecules per cubic inch. Thus far, it has been impossible to completely guard against the contamination of other specimens through contact with these airborne copies, regardless of the degree of care exercised by the operator(s).
The following techniques for dealing with the contamination problems created by amplification of target sequences, although suggested for use in a PCR type of amplification procedure, would be generally applicable to other types of amplification procedures: (1) physical separation (such as separate rooms) of pre-amplification and post-amplification samples; (2) separate storage and aliquotting of reagents; (3) the use of positive displacement pipettes; (4) meticulous laboratory technique; and, (5) cautious selection of controls. Amplifications--A forum for PCR Users, 2, 4 (1989). These measures, however, are not only costly, but assume the most ideal of conditions. Furthermore, even if these expensive techniques are practiced fastidiously, precautions such as these cannot completely eliminate the contamination problem. There remain inherent difficulties from laboratory workers who invariably carry contamination on their bodies and clothes and from the circulation of contaminated air from room to room through air vents. Kitchin, Nature, 344, 201 (1990).
Treatment of reagents with ultraviolet light has been suggested for the control of contaminant amplification product in a PCR type of amplification procedure. This suggestion is based on the known ability of ultraviolet light to destroy the integrity of DNA. Although the mechanism for this action is unknown, it has been demonstrated that PCR-based contaminant amplification product can be destroyed in buffers as well as in primer, dNTP, and Taq polymerase preparations by irradiation with ultraviolet light. Sarkar et al, Nature, 343, 27 (1990). Although the single-stranded PCR primers are apparently able to survive this treatment, double-stranded pairs of LCR probes are likely to be more sensitive to the irradiation treatment, and may be destroyed. Furthermore, irradiation with ultraviolet light cannot be used directly on unassayed test samples, because this would result in the destruction of double-stranded target molecules by the irradiation treatment.
It is an objective of the present invention to provide a cost effective method for significantly reducing the carryover contamination encountered when using amplification procedures in diagnostic probe assays. It is a further objective of the present invention to provide a contamination reduction method which is simple to perform and which is adaptable to a number of different types of amplification procedures.